biochemical characterization of lnr_a of human notch1 and notch2
DESCRIPTION
Biochemical Characterization of LNR_A of Human Notch1 and Notch2. Christina Hao. What is Notch?. Transmembrane protein receptors of 300-350kDa Highly conserved Regulates cell growth, differentiation, and cell death in a vast array of tissues through Notch signaling pathway - PowerPoint PPT PresentationTRANSCRIPT
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Biochemical Characterization of LNR_A of Human Notch1 and Notch2
Christina Hao
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What is Notch?
Transmembrane protein receptors of 300-350kDa
Highly conserved
Regulates cell growth, differentiation, and cell death in a vast array of tissues through Notch signaling pathway
Deregulation of Notch signaling pathway is associated with diseases, eg. Cancer
Four mammalian Notch homologs identified (Notch 1-4)
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Notch Signaling Pathway
Receiving Cell
Signaling Cell
Ligand
A B C ICN
Ligand-binding Region
Negative regulatory region (NRR)
HD Domain
LNR Domain
A B C HD-N HD-C
S2 S3
Nucleus
Notch Activation I. Ligand binding
II. Regulated cleavages
III. Release of intracellular notch/ Regulation of gene transcription
S1
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Structural View of NRR
A B C HD-N HD-C ICN
Negative regulatory region (NRR)
S1 S3S2LNRs are important for maintaining the receptor in
its resting conformation prior to ligand binding.
A B C HD-N HD-C
S1 S2 S3
EGF-like Repeats
Gordon, Vardar-Ulu, Histen, Sanchez-Iriarry, Aster, Blacklow (2007) Structural basis for autoinhibition of Notch. Nature: Structural and molecular biology
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Biochemical Characterization of LNR_A in Human Notch1 and Notch2 Lin12/Notch repeats are structurally independent,
disulfide-rich, protein modules of 35 residues. Can be biochemically
characterized in vitro
Requires large amount of proteins for characterization Goal: Optimize protein production in E.coli expr
ession system. Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules fro
m Human Notch1. American Chemical Society (42)7061-7067
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Research Protocol: Optimizing Recombinant Protein
Expression in Escherichia Coli
CaCl2 competent cells
1 Transformation
Monitor optical density
Inoculate culture with single colony
Grow at 37o C
Collect hourly Samples for
4 hours
Induce with 0.5, 0.1 mM IPTG at 0.5 and0.8 OD
2
34
566x His tag Nickel
affinity chromatography
7
Run Gel
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Cell Lines
Protocol Overview:
Competent cells :::::Transformation :::::Inoculation :::::Induction :::::Purification :::::Gel
Cell lines Main Features
BL21 (DE3)
T7 polymerase Lacks two enzymes
BL21 (DE3)-PlysS
T7 polymerase Lacks two enzymes T7 lysozyme
BL21
(DE3)-
RIPL
T7 polymerase Lacks two enzymes Carry extra genes that recognize mammalian arginine, isoleucine and leucine condons
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Protocol Overview:
Competent cells :::::Transformation :::::Inoculation :::::Induction :::::Purification :::::Gel
Origin
T7 promoterTarget gene
T7 terminator His-Tag
Lac I
Vector: pET15
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Induction: IPTGProtocol
Overview:
Competent cells
:::::Transformation :::::Inoculation :::::Induction :::::Purification :::::Gel
Repressorlac 1
T7 RNA polymerase
Lac Operon
E coli
mRNA
T7 Promotor Operator
Target genes
IPTG
IPTG
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Protocol Overview:
Competent cells
:::::Transformation :::::Inoculation :::::Induction :::::Purification :::::
Gel
Results Expected outcome:
Molecular
Weight Uninduced 1 hr. 2 hr.3 hr. 4 hr.
6kda
6kDa
Where are the proteins?
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Conclusion:
No significant production of hNotch1 LNR_A was present in E. coli under these experimental parameters:
DE3, plysS, RIPL host strains with pET15 vector grown at 37o C and induced with 0.1, 0.5mM IPTG at 0.5, 0.8OD.
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DiscussionPossible reasons for low expression of target protein: Rapid proteolytic degradation Decreased mRNA stability Toxicity upon induction Unsuitable expression system
What is next: Continue experimenting with different conditions (eg. te
mperature, media contents, etc.) difficult for drastic improvement
Inclusion bodies has proven to work previously
mRNA
T7 RNA polymerase
E. coli
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Future Projects
Determine the Ca2+ affinity of LNRs for other all notch via Isothermal Titration Calorimetry
Test different metals
Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules from Human Notch1. American Chemical Society (42)7061-7067
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Impact of Proposed Projects
Information acquired through these studies will:
Facilitate the development of structural and functional hypotheses about the regulation of Notch signaling
Provide insight into how failure in tight regulation can lead to disease states
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References
Gordon, Vardar-Ulu, Histen, Sanchez-Iriarry, Aster, Blacklow (2007) Structural basis for autoinhibition of Notch. Nature: Structural and molecular biology
Sjolund, Manetopoulos, Stockhausen, Axelson (2005). Review: The Notch pathway in cancer: Differentiation gone awry. European Journal of Cancer 41: 2620-2629
Sorensen, Mortensen (2004) Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology (115) 2:113-128
Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules from Human Notch1. American Chemical Society (42)7061-7067
http://www.emdbiosciences.com/product/69661 http://wolfson.huji.ac.il/expression/Bacterial_Strains.htm#strains-exp
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Acknowledgements
Didem Vardar-Ulu Sharline Madera
Mentoring in the Science Program Fund
Rhulman
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Questions?
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Thank You